Zinc—amino acid—lauryl sulfate complex with antimicrobial activity

ABSTRACT

Described herein are zinc-amino acid-lauryl sulfate complexes and oral care compositions comprising the same; and methods of making and using the same are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/749,714, filed on Oct. 24, 2018.

BACKGROUND

Zinc and basic amino acids are known to have several benefits and arecurrently used as active materials in oral care compositions. Zinc hasbeen shown to have antibacterial properties in plaque and cariesstudies. Arginine and other basic amino acids have benefits in combatingcavity formation and tooth sensitivity. Arginine was initiallyintroduced as an additive to oral care compositions for the treatment oftooth sensitivity. When combined with calcium, arginine has beenclinically proven to be effective in treating dentinal sensitivity byplugging and sealing dentinal tubules. Basic amino acids are also knownto have significant anti-caries benefits.

Zinc-amino acid complex forms a soluble cationic moiety, which in turnmay form a salt with a halide or other anion. A zinc-lysine complex(“ZLC”) having the chemical structure [Zn(C₆H₁₄N₂O₂)₂Cl]⁺Cl⁻ has beendescribed in prior art (e.g., WO 2014/098813). When placed in an oralcare formulation, this complex provides an effective concentration ofzinc ions to the enamel, thereby protecting against erosion, reducingbacterial colonization and biofilm development, and providing enhancedshine to the teeth (WO 2014/098824). Moreover, upon use, the formulationprovides a precipitate that can plug the dentinal tubules, therebyreducing the sensitivity of the teeth.

In oral care formulations containing zinc and amino acids, otheringredients present in the formulations can potentially interact withzinc and amino acids to form new species in the final product and affecttheir performance. The present invention relates to the identificationof new complexes formed by the interaction of zinc-amino acid complexesand sodium lauryl sulfate.

BRIEF SUMMARY

The present invention describes zinc-amino acid-lauryl sulfatecomplexes. In some embodiments, the amino acid in the complex is a basicamino acid. In a preferred embodiment, the zinc-basic amino acid-laurylsulfate complex is a zinc-arginine (Arg)-lauryl sulfate (LS) orzinc-lysine (Lys)-lauryl sulfate having the chemical structure[Zn(Arg)₂](LS)₂ or [Zn(Lys)₂](LS)₂. In an embodiment, the zinc-aminoacid-lauryl sulfate complex is in powder form.

The present invention also provides methods of producing a zinc-basicamino acid-lauryl sulfate complex by combining sodium lauryl sulfate anda zinc-amino acid complex in an aqueous solution, e.g., an aqueoussolution having pH of from 6 to 9, e.g., from 6.5-8.5, or from 7 to 8.In some embodiments, the method further comprises a step of filteringand drying the zinc-amino acid-lauryl sulfate complex.

The present invention also provides oral care compositions comprising azinc-amino acid-lauryl sulfate complex, e.g., zinc-basic aminoacid-lauryl sulfate complex, e.g., zinc-arginine-lauryl sulfate orzinc-lysine-lauryl sulfate. In some embodiments, the zinc-aminoacid-lauryl sulfate complex is added to the composition as a preformedcomplex. In some embodiments, the zinc-amino acid-lauryl sulfate complexmay be present in an amount of 0.1-10% by weight of the composition.

The present invention also provides use of a zinc-amino acid-laurylsulfate complex in powder form for the manufacture of an oral carecomposition for reducing and inhibiting acid erosion of the enamel,cleaning the teeth, reducing bacterially-generated biofilm and plaque,reducing gingivitis, inhibiting tooth decay and formation of cavities,and reducing dentinal hypersensitivity.

Further areas of applicability of the present disclosure will becomeapparent from the detailed description provided hereinafter. It shouldbe understood that the detailed description and specific examples, whileindicating the preferred embodiment of the disclosure, are intended forpurposes of illustration only and are not intended to limit the scope ofthe disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description and the accompanying drawings.

FIG. 1 shows the infrared spectra of sample 1A, sample 1B, L-arginine,and SLS powders. Shaded regions are examples of SLS and argininecorresponding bands that can be used for the identification of thesecomponents in sample 1A and 1B. Spectra are offset for clarity.

FIG. 2 shows ¹H NMR spectra of sample 1A, sample 1B, L-arginine, and SLSdissolved in deuterated methanol. The peaks are assigned to thecorresponding ¹H of LS and arginine.

FIG. 3 shows diffusion coefficient of arginine and LS in samples 1A and1B compared to that of pure arginine and SLS in methanol at 25° C.

FIG. 4A shows the mass spectrum of sample 1A at 410.7/412.7/414.7 undera positive detection mode. The triplet peak is typical of zinc arginate.FIG. 4B shows the zinc arginate structure and the calculated m/z Dalton.

FIG. 5 shows the mass spectrum of sample 1A under a negative detectionmode.

FIG. 6 shows the infrared spectra of samples 2A, 2B and 2C vs. SLS andL-Lysine powders. Shaded regions are examples of SLS and lysineassociated bands that can be used for the identification of thesecomponents in samples 2A, 2B and 2C, SLS and Lysine spectra are offsetfor clarity.

FIG. 7 shows ¹H NMR spectra of samples 2A, 2B and 2C, SLS and L-Lysinein d-methanol and their peak assignments.

FIG. 8 shows the mass spectrum of samples 2A, 2B and 2C in methanolunder a positive detection mode.

FIG. 9 shows the mass spectrum of samples 2A, 2B and 2C in methanolunder a negative detection mode.

FIGS. 10A, B and C show experimental potentiometric titration curves(circles) and fits (solid lines) for the zinc, lysine, SLS systems([Zn]: [Lys]: [SLS]=1:1:5 (FIG. 10A), 1:1:10 (FIG. 10B) or 1:1:20 (FIG.10C)). FIG. 10D shows speciation diagram for the zinc, lysine, SLSsystem at 0.2M ionic strength and pH=8 as a function of SLSconcentration.

DETAILED DESCRIPTION

The following description of the preferred embodiment(s) is merelyexemplary in nature and is in no way intended to limit the disclosure,its application, or uses.

As used throughout, ranges are used as shorthand for describing each andevery value that is within the range. Any value within the range can beselected as the terminus of the range. In addition, all references citedherein are hereby incorporated by referenced in their entireties. In theevent of a conflict in a definition in the present disclosure and thatof a cited reference, the present disclosure controls.

As used herein, the term “preformed complex” means that the zinc-aminoacid-lauryl sulfate complex is not formed in situ in the oral carecomposition, e.g. through the reaction of zinc, amino acid and laurylsulfate.

Unless otherwise specified, all percentages and amounts expressed hereinand elsewhere in the specification should be understood to refer topercentages by weight. The amounts given are based on the active weightof the material.

The present invention relates to zinc-amino acid-lauryl sulfatecomplexes. Some commercialized oral care products contain zinc-aminoacid complexes (e.g., ZLC) or contain both zinc ion sources (e.g., zincoxide or zinc citrate) and amino acids (e.g., lysine or arginine). Zincand amino acid can potentially interact with other ingredients presentin the oral care products to form a new compound, thus affecting thestability of zinc, amino acid or zinc-amino acid complex in the product.The inventors have found that zinc-amino acid complexes (e.g., ZLC andzinc arginate) interact with sodium lauryl sulfate, a surfactant thatalso possesses antibacterial activity, to form water insolublezinc-amino acid-lauryl sulfate complexes. In contrast to ZLC and zincarginate [Zn(Arg)₂]²⁺, the zinc-amino acid-lauryl sulfate complexes areinsoluble in water. Thus, the zinc-amino acid-lauryl sulfate complexesof the present invention offer opportunities to provide slow release ofthree active materials (zinc, amino acid and lauryl sulfate), whileavoiding the unpleasant metallic taste and aftertaste of soluble zinccompounds. The water-insoluble zinc-amino acid-lauryl sulfate complexesmay also occlude dentin tubules. Thus, the zinc-amino acid-laurylsulfate complexes can be used as a dentin tubule occlusion agent in oralcare products. In addition, it has been found that the zinc-aminoacid-lauryl sulfate complexes of the present invention have betterantibacterial activity compared to corresponding zinc-amino acidcomplexes (e.g., ZLC and zinc arginate). Since the zinc-aminoacid-lauryl sulfate complexes are insoluble upon preparation, they canbe prepared as a dry powder for use in manufacturing oral care productscomprising the zinc-amino acid-lauryl sulfate complexes. Use of thedried powder would allow savings on the quantities of starting materialsand overall liquid contents that need to be added into a product toachieve a given final concentration.

The invention provides, in one aspect, zinc-amino acid-lauryl sulfatecomplexes. The terms “zinc-amino acid-lauryl sulfate complex” or“zinc-amino acid-lauryl sulfate” as used herein refers to a waterinsoluble complex comprising zinc, amino acid and lauryl sulfate. Inthis disclosure, lauryl sulfate is sometimes referred to as “LS”. Thezinc-amino acid-lauryl sulfate complexes are formed by combining azinc-amino acid complex, e.g., ZLC or zinc arginate, and sodium laurylsulfate (SLS) in an aqueous solution. Commercial SLS products areusually a mixture of sodium alkyl sulfates with carbon chains of variouslengths, e.g., C₁₂-C₁₈, mainly lauryl. Therefore, the zinc-aminoacid-lauryl sulfate complex of the invention may contain a mixture ofalkyl sulfates with carbon chains of various lengths, e.g., C₁₂-C₁₈. Theterm “lauryl sulfate” or “LS” as used herein refers to an alkyl sulfatewith C₁₂ carbon chain or a mixture of alkyl sulfates with carbon chainsof various lengths, e.g., C₁₂-C₁₈ alkyl sulfates. In some embodiments,lauryl sulfate is a mixture of C₁₂-C₁₈ alkyl sulfates.

In some embodiments, the zinc-amino acid-lauryl sulfate complexes of thepresent invention have the chemical structure [Zn(amino acid)₂](LS)₂.The theoretical molar ratio of Zn:amino acid:LS in the complex is 1:2:2.However, the zinc-amino acid-lauryl sulfate complex of the presentinvention may comprise a small amount, e.g., less than 10%, less than5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or lessthan 0.01%, of impurities such as SLS (sodium lauryl sulfate), ZnO andZn(OH)₂.

In some embodiments, the zinc-amino acid-lauryl sulfate complexes of thepresent invention are in powder form.

Examples of amino acid in the zinc-amino acid-lauryl sulfate complex ofthe present invention include, but are not limited to, the commonnatural amino acids, e.g., lysine, arginine, histidine, glycine, serine,threonine, asparagine, glutamine, cysteine, selenocysteine, proline,alanine, valine, isoleucine, leucine, methionine, phenylalanine,tyrosine, tryptophan, aspartic acid, and glutamic acid. In preferredembodiments, the amino acid is a basic amino acid. The term “basic aminoacid” as used herein refers to naturally occurring basic amino acids,such as arginine, lysine, and histidine, as well as any basic amino acidhaving a carboxyl group and an amino group in the molecule, which iswater-soluble and provides an aqueous solution with a pH of about 7 orgreater. Accordingly, basic amino acids include, but are not limited to,arginine, lysine, citrulline, ornithine, creatine, histidine,diaminobutanoic acid, diaminopropionic acid, salts thereof orcombinations thereof. In some embodiments, the amino acid is lysine orarginine. In certain embodiments, the amino acid is lysine. In otherembodiments, the amino acid is arginine.

The zinc-amino acid-lauryl sulfate complexes of the present inventionmay be prepared by a process comprising combining sodium lauryl sulfateand a zinc-amino acid complex in an aqueous solution. In someembodiments, pH of the solution in which sodium lauryl sulfate and azinc-amino acid complex are combined is from 6 to 9, e.g. from 6.5-8.5or from 7 to 8. In other embodiments, pH of the solution in which sodiumlauryl sulfate and a zinc-amino acid complex are combined is notadjusted. In some embodiments, the prepared zinc-amino acid-laurylsulfate complexes may be further filtered and dried to be a powder.

In some embodiments, the zinc-amino acid complex that is combined withsodium lauryl sulfate is [Zn(Lys)₂Cl]⁺Cl⁻ (ZLC) or ([Zn(Arg)₂Cl]⁺Cl).These zinc-amino acid complexes can be prepared by combining ZnO,Lysine-HCl (or Arginine) and HCl in an aqueous solution, as described inWO 2014/098813. The formation of a white precipitate comprisingzinc-amino acid-lauryl sulfate complexes occurs immediately uponcombining sodium lauryl sulfate and the zinc-amino acid complex.

Alternatively, the zinc-amino acid complex that is combined with sodiumlauryl sulfate-lauryl sulfate may be prepared by combining a zinc ionsource and an amino acid in an aqueous solution. In some embodiments,the zinc ion source is a water soluble zinc ion source, e.g., zincchloride. In some embodiments, the amino acid is a basic amino acid,e.g., arginine or lysine. In some embodiments, pH of the solution inwhich the zinc ion source and the amino acid are combined is from 6 to9, e.g. from 6.5-8.5 or from 7 to 8. In other embodiments, pH of thesolution in which sodium lauryl sulfate and a zinc-amino acid complexare combined is not adjusted. The formation of a white precipitatecomprising zinc-amino acid-lauryl sulfate complexes occurs immediatelyupon adding sodium lauryl sulfate to the zinc-amino acid complexsolution prepared by combining a zinc ion source and an amino acid in anaqueous solution.

The invention thus provides, in one aspect, zinc-amino acid-laurylsulfate complexes (Compound 1).

In various aspects, Compound 1 included the following:

-   -   1.1 Compound 1, wherein the zinc-amino acid-lauryl sulfate has        the chemical structure [Zn(amino acid)₂](LS)₂.    -   1.2 Compound 1 or 1.1, wherein the zinc-amino acid-lauryl        sulfate is insoluble in an aqueous solution, e.g. in water at pH        7-8, e.g., wherein the zinc-amino acid-lauryl sulfate        precipitates in an aqueous solution, e.g. in water at pH 7-8.    -   1.3 Any foregoing compound, wherein the zinc-amino acid-lauryl        sulfate is a zinc-basic amino acid-lauryl sulfate.    -   1.4 Compound 1.3, wherein the amino acid is arginine or lysine.    -   1.5 Compound 1.4, wherein the amino acid is arginine.    -   1.6 Compound 1.5, wherein the zinc-amino acid-lauryl sulfate has        the chemical structure [Zn(Arg)₂](LS)₂.    -   1.7 Compound 1.4, wherein the amino acid is lysine.    -   1.8 Compound 1.7, wherein the zinc-amino acid-lauryl sulfate has        the chemical structure [Zn(Lys)₂](LS)₂.    -   1.9 Any foregoing compound, wherein the zinc-amino acid-lauryl        sulfate is in powder form.    -   1.10 Any foregoing compound, wherein the zinc-amino acid-lauryl        sulfate is prepared by a process comprising combining sodium        lauryl sulfate and a zinc-amino acid complex in an aqueous        solution.    -   1.11 Compound 1.10, wherein the zinc-amino acid complex is a        zinc-basic amino acid complex, e.g., [Zn(Lys)₂Cl]⁺Cl⁻(ZLC) or        [Zn(Arg)₂Cl]⁺Cl⁻.    -   1.12 Compound 1.10, wherein the zinc-amino acid complex is        prepared by combining a zinc ion source and an amino acid in an        aqueous solution.    -   1.13 Compound 1.12, wherein the amino acid is a basic amino        acid, e.g., arginine or lysine.    -   1.14 Compound 1.12 or 13, wherein the zinc ion source is a water        soluble zinc salt, e.g., zinc chloride.    -   1.15 Any foregoing compound, wherein the lauryl sulfate is a        mixture of C₁₂-C₁₈ alkyl sulfates.

The invention provides, in another aspect, methods (Method 2) for theproduction of zinc-amino acid-lauryl sulfate complexes (any of Compounds1, et. seq.), comprising combining sodium lauryl sulfate and azinc-amino acid complex in an aqueous solution, e.g. water.

In various aspects, Compound 1 included the following:

-   -   2.1 Method 2, wherein pH of the solution in which sodium lauryl        sulfate and the zinc-amino acid complex is combined is from 6 to        9, e.g. from 6.5 to 8.5 or from 7 to 8.    -   2.2 Method 2, wherein pH of the solution in which sodium lauryl        sulfate and the zinc-amino acid complex is not adjusted.    -   2.3 Any foregoing method, wherein the zinc-amino acid-lauryl        sulfate is a zinc-basic amino acid-lauryl sulfate.    -   2.4 Method 2.3, wherein the zinc-amino acid complex is        [Zn(Lys)₂Cl]⁺Cl⁻ (ZLC) or [Zn(Arg)₂Cl]⁺Cl⁻.    -   2.5 Method 2.3, wherein the zinc-amino acid complex is prepared        by combining a soluble zinc salt, e.g., zinc chloride, and a        basic amino acid, e.g., arginine or lysine, in an aqueous        solution, e.g. water.    -   2.6 Method 2.5, wherein pH of the solution in which the sodium        zinc salt and the basic amino acid is combined is from 6 to 9,        e.g., from 6.5 to 8.5 or from 7 to 8.    -   2.7 Method 2.5, wherein pH of the solution in which the sodium        zinc salt and the basic amino acid is not adjusted.    -   2.8 Any foregoing method, wherein the method further comprises a        step of filtering and drying the zinc-amino acid-lauryl sulfate        complex.    -   2.9 Any foregoing method, wherein the sodium lauryl sulfate is a        mixture of sodium C₁₂-C₁₈ alkyl sulfates.

The invention provides, in another aspect, oral care compositions(Composition 3) comprising zinc-amino acid-lauryl sulfate complexes (anyof Compounds 1, et. seq.), wherein the zinc-amino acid-lauryl sulfatecomplex is added to the composition as a preformed complex.

In various aspects, Composition 3 includes:

-   -   3.1. Composition 3, wherein the zinc-amino acid-lauryl sulfate        is present in an amount of from 0.1 to 10%, from 0.5 to 10%,        from 1 to 10%, from 2 to 10%, from 2 to 8%, from 2% to 4%, from        4% to 6%, from 6% to 8%, from 8% to 10%, or from 5% to 6% by        weight of the composition.    -   3.2. Composition 3 or 3.1, wherein the oral care composition is        a dentifrice.    -   3.3. Any foregoing composition, wherein the zinc-amino        acid-lauryl sulfate is added to the composition in powder form.    -   3.4. Any foregoing composition, wherein the pH of the        composition is 4.5-10.5, e.g., 6-9, 6.5-8.5, 7.0-8.5, 7.0-8.0,        7.5-8.0, or 8.0-8.5.    -   3.5. Any foregoing composition, wherein the composition        comprises one or more soluble phosphate salts, e.g. selected        from tetrasodium pyrophosphate (TSPP), sodium tripolyphosphate        (STPP) and combinations thereof.    -   3.6. Any foregoing composition, wherein the composition        comprises an effective amount of a fluoride ion source, e.g.,        providing 500 to 3000 ppm fluoride.    -   3.7. Composition 3.6, wherein the fluoride is a salt selected        from stannous fluoride, sodium fluoride, potassium fluoride,        sodium monofluorophosphate, sodium fluorosilicate, ammonium        fluorosilicate, amine fluoride (e.g.,        N′-octadecyltrimethylendiamine-N,N,N′-tris(2-ethanol)-dihydrofluoride),        ammonium fluoride, titanium fluoride, hexafluorosulfate, and        combinations thereof.    -   3.8. Any foregoing composition, wherein the composition        comprises a humectant, e.g., selected from glycerin, sorbitol,        propylene glycol, polyethylene glycol, xylitol, and mixtures        thereof, e.g., comprising at least 30%, e.g., 30-50% glycerin,        by weight of the composition.    -   3.9. Any foregoing composition, wherein composition further        comprises one or more surfactants, e.g., selected from anionic,        cationic, zwitterionic, and nonionic surfactants, and mixtures        thereof, e.g., in an amount of from 0.01% to 5%, from 0.01% to        2%, from 1% to 2%, or about 1.5%, by weight of the composition.    -   3.10. Any foregoing composition, wherein the composition further        comprises an anionic surfactant, e.g., a surfactant selected        from sodium lauryl sulfate, sodium ether lauryl sulfate, and        mixtures thereof, e.g., in an amount of from about 0.3% to about        4.5% by weight, 1-2%, or about 1.5% by weight of the        composition.    -   3.11. Any foregoing composition, wherein the composition        comprises a zwitterionic surfactant, for example a betaine        surfactant, for example cocamidopropylbetaine, e.g. in an amount        of 0.1%-4.5% by weight, e.g., 0.5-2% cocamidopropylbetaine by        weight of the composition    -   3.12. Any foregoing composition, wherein the composition        comprises gum strips or fragments.    -   3.13. Any foregoing composition, wherein the composition        comprises flavoring, fragrance and/or coloring.    -   3.14. Any foregoing composition, wherein the composition        comprises an effective amount of one or more antibacterial        agents in addition to the zinc-amino acid-lauryl sulfate        complex, for example comprising an antibacterial agent selected        from halogenated diphenyl ether (e.g. triclosan), herbal        extracts and essential oils (e.g., rosemary extract, tea        extract, magnolia extract, thymol, menthol, eucalyptol,        geraniol, carvacrol, citral, honokiol, catechol, methyl        salicylate, epigallocatechin gallate, epigallocatechin, gallic        acid, miswak extract, sea-buckthorn extract), bisguanide        antiseptics (e.g., chlorhexidine, alexidine or octenidine),        quaternary ammonium compounds (e.g., cetylpyridinium chloride        (CPC), benzalkonium chloride, tetradecylpyridinium chloride        (TPC), N-tetradecyl-4-ethylpyridinium chloride (TDEPC)),        phenolic antiseptics, hexetidine, octenidine, sanguinarine,        povidone iodine, delmopinol, salifluor, metal ions (e.g., zinc        salts, for example, zinc citrate, stannous salts, copper salts,        iron salts), sanguinarine, propolis and oxygenating agents        (e.g., hydrogen peroxide, buffered sodium peroxyborate or        peroxycarbonate), phthalic acid and its salts, monoperthalic        acid and its salts and esters, ascorbyl stearate, oleoyl        sarcosine, alkyl sulfate, dioctyl sulfosuccinate,        salicylanilide, domiphen bromide, delmopinol, octapinol and        other piperidino derivatives, nicin preparations, chlorite        salts; and mixtures of any of the foregoing; e.g., comprising        triclosan or cetylpyridinium chloride.    -   3.15. Any foregoing composition, wherein the composition        comprises an abrasive.    -   3.16. Composition 3.15, wherein the abrasive is selected from        silica abrasives, calcium phosphate abrasives, e.g., tricalcium        phosphate (Ca₃(PO₄)₂), hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), or        dicalcium phosphate dihydrate (CaHPO₄ •2H₂O, also sometimes        referred to herein as DiCal) or calcium pyrophosphate; calcium        carbonate abrasive; or abrasives such as sodium metaphosphate,        potassium metaphosphate, aluminum silicate, calcined alumina,        bentonite or other siliceous materials, and combinations        thereof.    -   3.17. Any foregoing composition, wherein the composition        comprises a whitening agent, e.g., a selected from the group        consisting of peroxides, metal chlorites, perborates,        percarbonates, peroxyacids, hypochlorites, and combinations        thereof.    -   3.18. Any foregoing composition, wherein the composition        comprises hydrogen peroxide or a hydrogen peroxide source, e.g.,        urea peroxide or a peroxide salt or complex (e.g., such as        peroxyphosphate, peroxycarbonate, perborate, peroxysilicate, or        persulphate salts; for example calcium peroxyphosphate, sodium        perborate, sodium carbonate peroxide, sodium peroxyphosphate,        and potassium persulfate);    -   3.19. Any foregoing composition, wherein the composition        comprises an agent that interferes with or prevents bacterial        attachment, e.g., solbrol or chitosan.    -   3.20. Any foregoing composition, wherein the composition        comprises a soluble calcium salt, e.g., selected from calcium        sulfate, calcium chloride, calcium nitrate, calcium acetate,        calcium lactate, and combinations thereof.    -   3.21. Any foregoing composition, wherein the composition        comprises a physiologically or orally acceptable potassium salt,        e.g., potassium nitrate or potassium chloride, in an amount        effective to reduce dentinal sensitivity.    -   3.22. Any foregoing composition, wherein the composition        comprises a breath freshener, fragrance or flavoring.    -   3.23. Any foregoing composition for use to reduce and inhibit        acid erosion of the enamel, clean the teeth, reduce        bacterially-generated biofilm and plaque, reduce gingivitis,        inhibit tooth decay and formation of cavities, and reduce        dentinal hypersensitivity.

The oral care composition used in the present invention can be in theform of any oral care formulations, including dentifrice, toothpaste,gel, mouthwash, powder, cream, strip, gum, bead, film, floss or anyother known in the art. In some embodiments, the oral care compositionused in the present invention is a dentifrice.

The oral care compositions of the invention contain an orally acceptablecarrier. As used herein, an “orally acceptable carrier” refers to amaterial or combination of materials that are safe for use in thecompositions of the invention, commensurate with a reasonablebenefit/risk ratio. Such materials include but are not limited to, forexample, water, humectants, ionic active ingredients, buffering agents,anticalculus agents, abrasive polishing materials, peroxide sources,alkali metal bicarbonate salts, surfactants, titanium dioxide, coloringagents, flavor systems, sweetening agents, antimicrobial agents, herbalagents, desensitizing agents, stain reducing agents, and mixturesthereof. Such materials are well known in the art and are readily chosenby one skilled in the art based on the physical and aesthetic propertiesdesired for the compositions being prepared.

The oral care composition of the present invention may further comprisea basic amino acid, e.g., arginine, in addition to the basic amino acidpresent in zinc-amino acid-lauryl sulfate complexes. The basic aminoacids which can be used in the compositions include not only naturallyoccurring basic amino acids, such as arginine, lysine, and histidine,but also any basic amino acids having a carboxyl group and an aminogroup in the molecule, which are water-soluble and provide an aqueoussolution with a pH of about 7 or greater. Accordingly, basic amino acidsinclude, but are not limited to, arginine, lysine, citrullene,ornithine, creatine, histidine, diaminobutanoic acid, diamrinopropionicacid, salts thereof or combinations thereof. In a particular embodiment,the basic amino acids are selected from arginine, lysine, citrullene,and ornithine. In some embodiments, the basic amino acid is arginine,for example, L-arginine, or a salt thereof.

The oral care composition of the present invention may comprise dentintubule occlusion agents. Such dentin tubule occlusion agents include,but are not limited to, arginine-calcium carbonate complexes, silicas,polymethyl vinyl ether-maleic acid (PMV/MA) copolymers, oxalate salts,strontium salts, and combinations thereof.

The oral care composition of the present invention may includedesensitizing agents. Such desensitizing agents include, but are notlimited to, potassium salts such as potassium nitrate, potassiumbicarbonate, potassium chloride, potassium citrate, potassium tartrate,and potassium oxalate, capsaicin, eugenol, strontium salts, zinc salts,chloride salts, and combinations thereof. Such agents may be added ineffective amounts, which preferably vary between about 0.01% to about10% by weight based on the total weight of the composition, depending onthe agent chosen. In some embodiments, the desensitizing agent is apotassium salt in an amount of at least about 5% by weight of apotassium salt based on the total weight of the composition, e.g., fromabout 5% to about 10% by weight of a potassium salt based on the totalweight of the composition. In some embodiments, the desensitizing agentis potassium nitrate.

The oral care composition of the present invention may include one ormore fluoride ion sources, e.g., soluble fluoride salts. A wide varietyof fluoride ion-yielding materials can be employed as sources of solublefluoride in the present compositions. Representative fluoride ionsources include, but are not limited to, stannous fluoride, sodiumfluoride, potassium fluoride, sodium monofluorophosphate, sodiumfluorosilicate, ammonium fluorosilicate, amine fluoride, ammoniumfluoride, and combinations thereof. In certain embodiments, the fluorideion source includes stannous fluoride, sodium fluoride, sodiummonofluorophosphate as well as mixtures thereof. In certain embodiments,the oral care composition described herein may also contain a source offluoride ions or fluorine-providing ingredient in amounts sufficient tosupply about 25 ppm to about 25,000 ppm of fluoride ions, generally atleast about 500 ppm, e.g., about 500 to about 2000 ppm, e.g., about 1000to about 1600 ppm, e.g., about 1450 ppm. The appropriate level offluoride will depend on the particular application. A toothpaste forgeneral consumer use would typically have about 1000 to about 1500 ppm,with pediatric toothpaste having somewhat less. A dentifrice or coatingfor professional application could have as much as about 5,000 or evenabout 25,000 ppm fluoride. Fluoride ion sources may be added to thecompositions described herein at a level of about 0.01 wt. % to about 10wt. % in one embodiment or about 0.03 wt. % to about 5 wt. %, and inanother embodiment about 0.1 wt. % to about 1 wt. % by weight of thecomposition in another embodiment. Weights of fluoride salts to providethe appropriate level of fluoride ion will obviously vary based on theweight of the counter ion in the salt.

The oral care compositions of the present invention may include otheractive ingredients. The active ingredients include, for example, zincion sources, anti-bacterial active agents, anti-tartar agents,anti-caries agents, anti-inflammatory agents, anti-sensitivity agents,basic amino acids, e.g., arginine, enzymes, nutrients, and the like.Actives useful herein are optionally present in the compositions of thepresent invention in safe and effective amounts that are sufficient tohave the desired therapeutic or prophylactic effect in the human orlower animal subject to whom the active is administered, without undueadverse side effects (such as toxicity, irritation, or allergicresponse), commensurate with a reasonable risk/benefit ratio when usedin the manner of this invention. The specific safe and effective amountof the active will vary with such factors as the particular conditionbeing treated, the physical condition of the subject, the nature ofconcurrent therapy (if any), the specific active used, the specificdosage form, the carrier employed, and the desired dosage regimen.

One or more abrasive or polishing materials may also be included in theoral care composition of the present invention. The abrasive orpolishing material can be any material that is acceptable for use in adentifrice, does not excessively abrade dentin and is compatible withthe other components of the oral care composition. Exemplary abrasive orpolishing materials include, but are not limited to: silica abrasives,calcium phosphate abrasives, e.g., tricalcium phosphate (Ca₃(PO₄)₂),hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), or dicalcium phosphate dihydrate(CaHPO₄ •2H₂O, also sometimes referred to herein as DiCal) or calciumpyrophosphate; calcium carbonate abrasive; or abrasives such as sodiummetaphosphate, potassium metaphosphate, aluminum silicate, calcinedalumina, bentonite or other siliceous materials, and combinationsthereof.

The oral care composition of the present invention may include an agentto increase the amount of foam that is produced when the oral cavity isbrushed. Illustrative examples of agents that increase the amount offoam include, but are not limited to polyoxyethylene and certainpolymers including, but not limited to, alginate polymers. Thepolyoxyethylene may increase the amount of foam and the thickness of thefoam generated by the oral care carrier component of the composition.Polyoxyethylene is also commonly known as polyethylene glycol (“PEG”) orpolyethylene oxide. The polyoxyethylenes suitable for this compositionwill have a molecular weight of about 200,000 to about 7,000,000. In oneembodiment the molecular weight will be about 600,000 to about 2,000,000and in another embodiment about 800,000 to about 1,000,000. Polyox® isthe trade name for the high molecular weight polyoxyethylene produced byUnion Carbide. The polyoxyethylene may be present in an amount of about1% to about 90%, in one embodiment about 5% to about 50% and in anotherembodiment about 10% to about 20% by weight of the oral care carriercomponent of the oral care compositions. Where present, the amount offoaming agent in the oral care composition (i.e., a single dose) isabout 0.01 to about 0.9% by weight, about 0.05 to about 0.5% by weight,and in another embodiment about 0.1 to about 0.2% by weight.

The oral care compositions of the present invention may further includeat least one surfactant or solubilizer in addition to lauryl sulfatethat may be released from zinc-amino acid-lauryl sulfate complexes ofthe invention. Suitable surfactants include neutral surfactants (such aspolyoxyethylene hydrogenated castor oil or fatty acids of sugars),anionic surfactants (such as sodium lauryl sulfate), cationicsurfactants (such as the ammonium cation surfactants) or zwitterionicsurfactants. These surfactants or solubilizers may be present in amountsof typically from 0.01% to 5%; from 0.01% to 2%; or from 1% to 2%; orabout 1.5%, by weight of the composition. These amounts do not includelauryl sulfate that may be released from zinc-amino acid-lauryl sulfatecomplexes of the invention. Thus, the actual amount of surfactants orsolubilizers in the oral care composition may be higher.

The oral care compositions of the present invention may include one ormore humectants. Humectants can reduce evaporation and also contributetowards preservation by lowering water activity, and can also impartdesirable sweetness or flavor to compositions. Suitable humectantsinclude edible polyhydric alcohols such as glycerin, sorbitol, xylitol,propylene glycol as well as other polyols and mixtures of thesehumectants. Other useful materials may also include orally acceptablealcohols, or polymers, e.g., such as polyvinylmethyl ether maleic acidcopolymers, polysaccharides (e.g. cellulose derivatives, for examplecarboxymethyl cellulose, or polysaccharide gums, for example xanthan gumor carrageenan gum). In some embodiments, the humectant can be presentin an amount of from 20% to 60%, for example from 30% to 50%, forexample from 40% to 45%, by weight of the composition.

The oral care compositions of the present invention may include apreservative. Suitable preservatives include, but are not limited to,sodium benzoate, potassium sorbate, methylisothiazolinone, parabenpreservatives, for example methyl p-hydroxybenzoate, propylp-hydroxybenzoate, and mixtures thereof.

The oral care compositions of the present invention may include asweetener such as, for example, saccharin, for example sodium saccharin,acesulfam, neotame, cyclamate or sucralose; natural high-intensitysweeteners such as thaumatin, stevioside or glycyrrhizin; or such assorbitol, xylitol, maltitol or mannitol. One or more of such sweetenersmay be present in an amount of from 0.005% to 5% by weight, for example0.01% to 1%, for example 0.01% to 0.5%, by weight of the composition.

The oral care compositions of the present invention may include aflavoring agent. Suitable flavoring agents include, but are not limitedto, essential oils and various flavoring aldehydes, esters, alcohols,and similar materials, as well as sweeteners such as sodium saccharin.Examples of the essential oils include oils of spearmint, peppermint,wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon,lemon, lime, grapefruit, and orange. Also useful are such chemicals asmenthol, carvone, and anethole. The flavoring agent is typicallyincorporated in the oral composition at a concentration of 0.01 to 3% byweight.

The invention further provides methods to reduce and inhibit aciderosion of the enamel, clean the teeth, reduce bacterially-generatedbiofilm and plaque, reduce gingivitis, inhibit tooth decay and formationof cavities, and reduce dentinal hypersensitivity, comprising applyingan effective amount of a composition of the invention, e.g., any ofComposition 3, et seq. to the teeth.

For example, in various embodiments, the invention provides methods to(i) reduce hypersensitivity of the teeth, (ii) reduce plaqueaccumulation, (iii) reduce or inhibit demineralization and promoteremineralization of the teeth, (iv) inhibit microbial biofilm formationin the oral cavity, (v) reduce or inhibit gingivitis, (vi) promotehealing of sores or cuts in the mouth, (vii) reduce levels of acidproducing bacteria, (viii) increase relative levels of non-cariogenicand/or non-plaque forming bacteria, (ix) reduce or inhibit formation ofdental caries, (x), reduce, repair or inhibit pre-carious lesions of theenamel, e.g., as detected by quantitative light-induced fluorescence(QLF) or electrical caries measurement (ECM), (xi) treat, relieve orreduce dry mouth, (xii) clean the teeth and oral cavity, (xiii) reduceerosion, (xiv) whiten teeth; (xv) reduce tartar build-up, and/or (xvi)promote systemic health, including cardiovascular health, e.g., byreducing potential for systemic infection via the oral tissues,comprising applying any of Compositions 3, et seq. as described above tothe oral cavity of a person in need thereof, e.g., one or more times perday. The invention further provides Compositions 3, et seq. for use inany of these methods.

The present invention further provides use of zinc-basic aminoacid-lauryl sulfate complexes (any of Compounds 1, et. seq.) in powderform for the manufacture of an oral care composition for reducing andinhibiting acid erosion of the enamel, cleaning the teeth, reducingbacterially-generated biofilm and plaque, reducing gingivitis,inhibiting tooth decay and formation of cavities, and reducing dentinalhypersensitivity.

EXAMPLES Example 1—Preparation and Characterization ofZinc-Arginine-Lauryl Sulfate Complex

Sample 1A: SLS powder was dissolved in 20 g of H₂O followed by pHadjustment with HCl to pH≈7. Arginine and ZnCl₂ were dissolved togetherin approximately 47.9 g of H₂O (Table 1) resulting in a clear solution.The pH of this mixture was adjusted with HCl to pH=7 followed by itsaddition to previously prepared SLS solution. A formation of whiteprecipitate occurred immediately. The precipitate was filtered, washedwith approximately 200 mL of deionized water and dried at roomtemperature.

Sample 1B: SLS powder was dissolved in 20 g of H₂O followed by pHadjustment with HCl to pH=8. Arginine and ZnCl₂ were dissolved togetherin approximately 47.9 g of H₂O (Table 1) resulting in a clear solution.The pH of this mixture was adjusted with HCl to pH=8 followed by itsaddition to previously prepared SLS solution. A formation of whiteprecipitate occurred immediately. The precipitate was filtered, washedwith approximately 200 mL of deionized water and dried at roomtemperature.

TABLE 1 Raw materials and their quantities used in preparation ofsamples 1A and 1B. Reagent H₂O (g) L-Arginine (g) ZnCl₂ Anhydrous (g)SLS (g) Sample 1A 67.91 3.86 1.03 5.14 Sample 1B 67.93 3.86 1.07 5.14

In addition to Samples 1A and 1B, control solutions without ZnCl₂ andwithout arginine were also prepared. The ratios between ingredients werekept the same as shown in Table 1. Arginine-SLS solutions at pH=7 and 8remained clear. No visual signs of precipitation were apparentconfirming that zinc ion is needed for the precipitate formation tooccur. The combination of ZnCl₂ and SLS resulted in white precipitate atpH>6 due to the Zn-LS interaction/precipitation and competing reactionof zinc hydroxide formation.

To characterize the composition of the precipitate formed uponcombination of ZnCl₂, arginine and SLS, infrared spectroscopic analysiswas performed. Infrared spectra were collected using a Bruker Vertex 70FTIR spectrometer (Bruker Optics, Billerica, Mass.) equipped with aGladiATR diamond ATR accessory (Pike technologies, Madison, Wis.). Thespectral range was 80-4000 cm⁻¹ and a resolution of 4 cm⁻¹ was used. Allmeasurements were carried out at room temperature. Infrared spectra areshown in FIG. 1. The vibrational spectra of samples 1A and 1B werecompared with the pure arginine and SLS absorption profiles. Clearfingerprints of arginine and SLS are apparent in the spectra of samples1A and 1B. For example, the band near 1200 cm⁻¹ that corresponds to theSO₂ asymmetric vibration (v_(as)(SO₂)) is clearly observed in sample 1Aand 1B (Viana et al. (2012), Adv. Phys. Chem., 2012, 1-14). Strong C—Hstretching modes (v_(as)(CH)) near 2900 cm⁻¹ serve as another distinctindicator of the lauryl sulfate component. A cluster of bands near 1600cm⁻¹ is a unique fingerprint of arginine that arises due to acombination of bending modes of amino group and stretching vibrations ofcarboxylate and guanidinium groups (Barth (2000), Prog. Biophys. Mol.Biol., 74, 141-173; Hernández et al. (2010), J. Phys. Chem. B 114 (2),1077-1088). Presence of both components, arginine and LS in the solidphase confirms that the major component of the precipitate in samples 1Aand 1B cannot be attributed to zinc hydroxide or zinc lauryl sulfateformation. Importantly, the bands of arginine and lauryl sulfate insamples 1A and 1B are substantially different in their intensity,shape/width profile and peak positions from those of pure arginine andSLS compounds. This indicates modifications to their local structure dueto interaction, which cannot be achieved just from a physical mixture ofcompounds. FTIR spectra of samples 1A and 1B are practically identical,suggesting their similar local structure and no significant effect of pHon complex formation, at least within pH 7-8 range.

To establish the ratio between zinc, arginine and lauryl sulfate as wellas to identify the presence of other potential ions in the samples,elemental analysis of Zn, N, S, Na, and Cl was performed following astandard protocol. The results of the analysis are shown in Table 2.

TABLE 2 Summary of elemental analysis for samples 1A and 1B. Molar RatioZn (%) N (%) S (%) Na (%) Cl (%) Arg/Zn LS/Zn Sample 6.18 10.32 5.280.00 — 1.95 1.74 1A Sample 6.21 10.38 6.15 0.00 0.00 1.95 2.02 1B Calc*6.92 11.86 6.79 — — 2 2 *Calculated values of Zn, N, and S forZn(Arg)₂(LS)₂

The experimental values in samples 1A and 1B are close to theoreticalvalues for Zn(Arg)₂(LS)₂. The lower experimental values compared totheoretical/calculated values might be due to the presence ofhydration/coordination water or residual moisture in the samples. Thesedata suggest that the precipitate formed upon combination of ZnCl₂,arginine and SLS is Zn(Arg)₂(LS)₂. The composition of Samples 1A and 1Bwas further determined by X-Ray photoelectron spectroscopy (XPS) using aPHI VersaProbe II Scanning XPS Microprobe (Ulvac PHI, Chanhassen,Minn.). Duplicate analyses of each sample were conducted. Both sampleswere analyzed as powders using a 200 micron analysis area. The elementalcomposition in atomic percent for each sample as well as the molepercent and molar ratio for arginine, lauryl sulfate, Zn and Na areshown in Table 3. The elemental compositions of samples 1A and 1B arethe same, indicating that pH has no significant effect on thecompositions of the precipitated solids. The Arg/Zn mole ratios forsamples 1A and 1B are consistent with that for Zn(Arg)₂(LS)₂. The Arg/LSand LS/Zn mole ratios for the samples differ from those ofZn(Arg)₂(LS)₂, indicating that residual SLS is present in the solid. Thedetection of Na also indicates that a small amount of SLS is present.When the moles of residual SLS are subtracted from the data, the molefractions for each sample match those for Zn(Arg)₂(LS)₂. Thus the XPSresults indicate that both samples are composed of Zn(Arg)₂(LS)₂, with aminor amount of residual SLS present in the solid.

TABLE 3 XPS analysis of samples 1A and 1B. Atomic Percent Sample C O NNa Zn Cl S Sample 1A 64.89 17.79 12.00 0.46 1.47 0.00 3.40 arg LS Zn Naarg/Zn arg/LS LS/Zn For Zn(Arg)₂(LS)₂ 2.0 1.0 2.0 Sample 1A 3.00 3.401.47 0.46 2.0 0.9 2.3 minus SLS 3.00 2.94 1.47 0.00 2.0 1.0 2.0 AtomicPercent Sample C O N Na Zn Cl S Sample 1B 64.50 17.75 12.37 0.40 1.550.00 3.45 arg LS Zn Na arg/Zn arg/LS LS/Zn For Zn(Arg)₂(LS)₂ 2.0 1.0 2.0Sample 1B 3.09 3.45 1.55 0.40 2.0 0.9 2.2 minus SLS 3.09 3.05 1.55 0.002.0 1.0 2.0

To understand the structure of the Zn(Arg)₂(LS)₂ complex, ¹H NMRmeasurements were performed on 1 wt % samples in deuterated methanolsolution. All NMR spectra were acquired on a Bruker Avance spectrometer(Bruker-Biospin, Billerica, Mass., USA) with a 5 mm BBI probe operatingat 500.0 MHz for ¹H at 25° C. Diffusion coefficients of molecules weremeasured by ¹H pulsed-field gradient NMR spectroscopy using an observedbroadband probe with a z axis gradient coil with maximal gradientstrength of 72 G/cm. A double stimulated echo pulse sequence withbipolar gradient pulses and two spoil gradients were used. The diffusiontime was 0.1 second. The duration of field gradient pulse was adjustedto be 4 milliseconds. The pulse gradients were incremented from 5 to 95%of the maximal gradient strength in a linear ramp with a total ofexperimental time of 30 minutes. FIG. 2 shows the ¹H NMR spectra ofsamples 1A and 1B as well as the spectra of pure L-arginine and SLS inmethanol. The NMR spectra of samples 1A and 1B were identical. Protonchemical shifts of LS remained constant in samples 1A and 1B, whereaschemical shifts of α, β, γ and δ protons in arginine show significantchanges in both samples compared to the pure compounds. This suggeststhat arginine coordinates to zinc in samples 1A and 1B, but LS does not.In addition, peak integrals of NMR spectra in samples 1A and 1B confirmthat the stoichiometric ratio between arginine and LS is 1:1.

To further confirm that arginine is bound to zinc in samples 1A and 1B,pulsed-field gradient NMR experiment was performed to measure thediffusion coefficient of arginine and LS in samples 1A and 1B inmethanol. FIG. 3 show that diffusion coefficient of arginine in bothsamples is reduced by a factor of about 45% compared to that of purearginine. In contrast, diffusion coefficients of LS free in solution andin samples 1A and 1B do not display significant changes. These resultsprovide strong evidence that arginine is bound to zinc, and LS is acounterion of the zinc-arginine complex in methanol.

To confirm the Zn(Arg)₂(LS)₂ complex structure, LC-MS analysis wasperformed on sample 1B using a AB Sciex tandem mass spectrometer (ABSciex LLC, Framingham, Mass., USA) equipped with an ESI interface andAgilent 1260 capillary LC system (Model Agilent 1260, AgilentTechnologies, Palo Alto, Calif., USA). The capillary LC system wasequipped with a capillary binary pump (Model G1376A), a DAD detector(G1315C), a micro vacuum degasser (Model G4225A), a thermostatted columncompartment (Model G1316A). The capillary pump was set under themicro-flow mode. The sample was directly introduced into MS detectorthrough a bypass injector. The flow rate was 70 μL/min and the injectedvolume was 5 μL. The AB Sciex tandem mass spectrometer was operated inthe positive-ion mode under the following conditions. Nitrogen (>99.99%)was used for curtain gas at 10 psi, ion source gas 1 and 2 at 10 and 10psi, respectively. ESI IonSpray voltage was set at 5.5 kV in ESIinterface for positive mode and 4.5 kV for a negative mode. Thedeclustering and entrance potential were set up at 80 and 5.5 v,respectively. The temperature of the ionization interface was maintainedat 350° C. The total ion count (TIC) mode was used for sample analysis,and the MS screen range was from 50 to 1500 m/z. Data was acquired withan Analyst software 1.6.2 system (AB Sciex LLC, Framingham, Mass., USA).FIGS. 4A and B indicate that one zinc ion is coordinated with twoarginine molecules and FIG. 5 shows the presence of free LS with chaindistribution. The four peaks with the different mass unit of 28 Daltonindicate the LS distribution with [(CH₂)₂] variation from C₁₂ to C₁₈. NoZn-Arg-LS complex was identified in either positive or negative mode onMS detector.

The presence of free LS in both NMR and MS data demonstrates that theZn(Arg)₂(LS)₂ compound dissociates in methanol. This suggests that LSmay act as a counterion to [Zn(Arg)₂]²⁺ complex with the overallcompound structure presented as [Zn(Arg)₂](LS)₂.

Geometry optimizations and harmonic frequency calculations wereperformed at the B3LYP/BS1 level in aqueous solution using the SMDsolvation model, BS1 designating a mixed basis set of SDD for zinc and6-31G(d,p) for other atoms. Because the M06 functional includesnoncovalent interactions and gives refined energies for metal-organiccomplexes, single-point energies were calculated for all of theB3LYP/BS1-optimized structures at the M06/BS2 level with solvationeffects modeled by SMD in aqueous solution, BS2 denoting a mixed basisset of SDD for zinc and 6-311++G(d,p) for other atoms. TheB3LYP/BS1-calculated harmonic frequencies were used to obtain zero-pointenergy-corrected Gibbs free energies at 298.15 K and 1 atm. The DensityFunctional Theory (DFT) calculation is shown below.[Zn(H₂O)₆]²⁺(aq)+2Ar(aq)→[Zn(Arg)₂]²⁺(aq)+6H₂O  (1) ΔG⁰=−53.2 kcal/molThe DFT result (ΔG: −53.2 kcal/mol) suggests that [Zn(Arg)₂]²⁺ is astable species existing in aqueous solution.

Example 2—Preparation and Characterization of Zinc-Lysine-Lauryl SulfateComplex

Three samples of zinc-lysine-lauryl sulfate complex were prepared inthis study. Samples 2A and 2B were prepared from the ZLC pre-mix and SLSusing the ratios of ZLC:SLS:H₂O as shown in Table 3. Sample 2C wasprepared from ZnCl₂+Lysine and SLS and was designed to mimic thepreparation of [Zn(Arg)₂](LS)₂ complex described in Example 1. Inaddition to samples 2A, 2B and 2C, a control solution (sample 2D)without zinc was also prepared. The Lysine-SLS solution at pH=8 remainedclear; no visual signs of precipitation were apparent confirming thatthe zinc ion is needed for the precipitate formation to occur.

TABLE 4 Raw materials and their quantities used in preparation ofsamples 2A, 2B, 2C, and 2D. Sample 2B Sample 2C Sample 2D Reagent (g)Sample 2A pH = 8 pH = 8 Control ZnO 2.10 2.10 — — ZnCl₂ — — 1.07 —Anhydrous L-Lysine-HCl 9.44 9.44 — — L-Lysine — — 3.24  3.24 HCl (conc.)1.37 1.36 — — (for ZLC pre-mix) H₂O 15.02  15.00  20.01  20.09 (for SLS)H₂O 14.99  16.51  47.89  47.84 (for Zn/Lys) SLS 3.49 3.60 5.14  5.15

Sample 2A (no pH adjustment): ZLC pre-mix was made by combining ZnO,L-Lysine-HCl, HCl (concentrated) and 15 g of H₂O (Table 4). The mixturewas stirred for one day to allow all of the ZnO to solubilize. SLSpowder was dissolved in a separate container in 15 g of H₂O. Once thefoam settled, the SLS solution was slowly added to the ZLC pre-mix underconstant stirring. The formation of a white precipitate occurredimmediately. The mixture was stirred for several hours and allowed toequilibrate for 1 day. The precipitate was filtered, washed withapproximately 250 mL of deionized water and dried at room temperature.

Sample 2B (pH=8): ZLC pre-mix was made by combining ZnO, L-Lysine-HCl,HCl (concentrated) and 16.5 g of H₂O (Table 4). The mixture was stirredfor one day to allow all of the ZnO to solubilize. SLS powder wasdissolved in a separate container in 15 g of H₂O and foam was allowed tosettle. Prior to combining ZLC and SLS, the pH of both solutions wasadjusted to pH=8. The ZLC pre-mix solution was filtered through the 0.45micron filter to remove zinc hydroxide partially formed at this pH. Uponcombination of ZLC and SLS the formation of a white precipitate occurredimmediately. The mixture was stirred for several hours and allowed toequilibrate for 1 day. The precipitate was filtered, washed withapproximately 250 mL of deionized water and dried at room temperature.

Sample 2C (pH=8): SLS powder was dissolved in 20 g of H₂O followed by pHadjustment with HCl to pH=8. Lysine and ZnCl₂ were dissolved together inapproximately 48 g of H₂O (Table 4) resulting in a clear solution. ThepH of this mixture was adjusted with HCl to pH=8 followed by itsaddition to the previously prepared SLS solution. The formation of awhite precipitate occurred immediately. The mixture was stirred forseveral hours and allowed to equilibrate for 1 day. The precipitate wasfiltered, washed with approximately 250 mL of deionized water and driedat room temperature.

Sample 2D (Control): SLS powder was dissolved in 20 g of H₂O followed bypH adjustment with HCl to pH=8. Lysine was dissolved in approximately 48g of H₂O (Table 4), and the pH was adjusted to pH=8. Addition of lysinesolution to SLS resulted in a clear solution with no sign of precipitateformation.

To characterize the composition of samples 2A, 2B and 2C, infraredspectroscopic analysis was performed as described in Example 1. FIG. 6shows the vibrational spectra of samples 2A, 2B and 2C as well the purearginine and SLS absorption profiles. Clear fingerprints of lysine andSLS are apparent in the spectra of samples 2A, 2B and 2C. For example,the band near 1200 cm⁻¹ that corresponds to the SO₂ asymmetric vibration(v_(as)(SO₂)) is clearly observed in samples 2A, 2B and 2C (Viana et al.(2012), Adv. Phys. Chem., 2012, 1-14). Strong C—H stretching modes(v_(as)(CH)) near 2900 cm⁻¹ serve as another distinct indicator of thelauryl sulfate component. A cluster of bands near 1600 cm⁻¹ is a uniquefingerprint of lysine that arises due to a combination of bending modesof amino groups and stretching vibration of the carboxylate group (Barth(2000), Prog. Biophys. Mol. Biol., 74, 141-173; Hernández et al. (2010),J. Phys. Chem. B 114 (2), 1077-1088). Presence of both components,lysine and LS in the solid phase confirms that the dominant component ofSamples 2A, 2B and 2C is not a result of zinc hydroxide and/or zinclauryl sulfate formation. Importantly, the bands of lysine and laurylsulfate in samples 2A, 2B and 2C are substantially different in theirintensity, shape/width profile and peak positions from those of the purelysine and SLS materials. This indicates modifications to the localstructure of the compounds due to chemical interaction, which cannot beachieved just from a physical mixture of two compounds. The FTIR spectraof samples 2A, 2B and 2C are alike, suggesting their similar localstructure and that there is no significant effect of pH or startingmaterials on the complex formation. A broad background in the lowfrequency range of sample 2B could be due to additional water/moisturepresence in this sample or due to an impurity that contributes to thebackground. Raman spectra of samples 2A, 2B and 2C were also found toclosely resemble each other, in agreement with the infrared data.

To establish the ratio between zinc, arginine and lauryl sulfate as wellas to identify the presence of other potential ions in the samples,elemental analysis of Zn, N, S, Na, and Cl was performed following astandard protocol. The results of the analysis are shown in Table 5.

TABLE 5 Summary of elemental analysis for samples 2A, 2B and 2C. MolarRatio Zn(%) N(%) S(%) Cl(%) Na(%) Lys/Zn LS/Zn LS/Lys Cl/Zn Sample 2A9.13 6.15 7.53 0.75 0.09 1.6 1.7 1.1 0.15 Sample 2B 12.47 5.41 7.47 0.180.05 1.0 1.2 1.2 0.03 Sample 2C 8.16 6.06 7.91 0.09 0.04 1.7 2.0 1.10.02 Calc* 7.36 6.3 7.22 0 0 2 2 1 0 Zn(Lys)₂(LS)₂ Calc** 9.93 8.5 9.745.38 0 2 1 0.5 1 Zn(Lys)₂Cl(LS) *Calculated values for Zn, N, and S,assuming Zn(Lys)₂(LS)₂ formula **Calculated values for Zn, N, S and Cl,assuming Zn(Lys)₂Cl(LS) formula

In Table 5, the experimental values in samples 2A, 2B and 2C arecompared with the calculated/theoretical values for two possibleformulas: (i) Zn(Lys)₂Cl(LS), and (ii) Zn(Lys)₂(LS)₂. In samples 2A, 2Band 2C, chlorine is present only in small quantities (likely animpurity) and therefore, Cl/Zn ratios are much less than 1, practicallyzero in samples 2B and 2C. This indicates that the Zn(Lys)₂Cl(LS)composition can be ruled out for the precipitates. The LS/Lys ratio forall three samples is close to 1, in a better agreement with theZn(Lys)₂(LS)₂ composition. For samples 2A and 2C the Lys/Zn and LS/Znratios are also closer to the theoretical composition of Zn(Lys)₂(LS)₂,although some excess of zinc is present in sample 2A. Similarly, sample2B displays an excess amount of zinc relative to Lysine and LS,resulting in substantially lower Lys/Zn and LS/Zn ratios. This suggeststhat some form of Zn(OH)₂ might be also formed at this pH and is presentin the precipitate. An alternative composition for instance,Zn(Lys)(LS)(OH) for sample 2B cannot be ruled out based on the elementalanalysis, but this is not supported by the FTIR/Raman data where allthree samples display similar spectral profiles suggesting similar localstructures.

The composition of samples 2A, 2B and 2C was further determined by XPS,which was performed as described in Example 1. Detected elements andtheir respective atomic percentages for each sample are shown in Table6. The percentages of the aliphatic (CH₂) and carboxylate C (COO⁻)functional groups are also indicated in Table 6. N was detected in bothcharged and uncharged chemical states and the percentages of both areshown in Table 6. As shown in Table 7, atomic ratios were alsocalculated from the compositional data to determine the stoichiometriesof the elements detected and compare those with the theoretical valuesfor Zn(Lys)₂(LS)₂.

TABLE 6 Compositions in atomic percent of samples 2A, 2B and 2Cdetermined by XPS. Atomic Percent Sample C_(total) CH₂ COO⁻ O N_(total)N N⁺ Zn S Na Sample 2A 69.82 51.81 3.75 18.35 6.75 3.23 3.52 1.70 3.390.00 Sample 2B 68.15 51.35 3.83 19.40 6.88 3.36 3.52 2.22 3.36 0.00Sample 2C 69.19 52.37 3.53 18.81 6.78 3.15 3.63 1.75 3.49 0.00Zn(Lys)₂(LS)₂ 65.45 50.91 3.64 21.82 7.27 3.64 3.64 1.82 3.64 0.00_((Theoretical))

TABLE 7 Atomic ratios for samples 2A, 2B and 2C determined by XPS.Atomic Ratio N_(total)/ COO/ COO/ N_(total)/ Sample C_(total) N⁺/NN_(total) N⁺ S N⁺/S N_(total)/Zn S/Zn Sample 2A 0.10 1.09 0.56 1.07 1.991.04 3.98 2.00 Sample 2B 0.10 1.05 0.56 1.09 2.05 1.05 3.11 1.51 Sample2C 0.10 1.15 0.52 0.97 1.95 1.04 3.89 2.00 Zn(Lys)₂(LS)₂ 0.11 1.00 0.501.00 2.00 1.00 3.99 2.00 _((Theoretical))

The compositions of all three samples were similar to the theoreticalcomposition for Zn(Lys)₂(LS)₂. The presence of C, O, N and S along withthe respective C and N functionalities clearly reflect the presence ofLys and LS in the samples. Zn was also observed in all three samples.However, the Zn concentration for sample 2B was slightly higher than thetheoretical value for Zn(Lys)₂(LS)₂. As shown in Table 7, the variousatomic ratios of organic components for each sample were in goodagreement with the theoretical values for Zn(Lys)₂(LS)₂. The resultsindicate that Lys and LS have a 1:1 stoichiometry, as predicted. Forsamples 2A and 2C, the N/Zn and S/Zn ratios were in excellent agreementwith the theoretical values for Zn(Lys)₂(LS)₂, and reflect the 2:1 Lysor LS to Zn stoichiometry. For sample 2B, the N/Zn and S/Zn ratios wereless than the theoretical values, indicating excess Zn relative to Lysand LS. The fact that both the N/Zn and S/Zn ratios are low may suggestthat a minor amount of ZnO or Zn(OH)₂ impurity is present in thissample. Overall, the XPS data are consistent with the elemental analysisresults and indicate that all three precipitates are composed ofZn(Lys)₂(LS)₂, with only sample 2B containing some Zn impurity.

XPS peak position data was further used to determine the chemicalbonding environment of elements in the samples. The XPS N, S and Zn peakdata for the three samples along with corresponding data for Lys and SLSare shown in Table 8. For all three samples, N is present in theuncharged (N) and positively charged (N⁺) chemical states. The N and N⁺peak positions for samples 2A, 2B and 2C differed from those for solidlysine indicating different N chemical bonding environments. In solidLys, the alpha C amine is positively charged, while the tail amine isuncharged (Williams et al. (2015), Angew. Chem. Int. Ed., 54 (13),3973-7). In Zn(Lys)₂(LS)₂, the alpha C amine is coordinated to Zn andwould not be charged but the tail amine is positively charged andpresumably coordinated to LS. These differences in bonding between Lysand Zn(Lys)₂(LS)₂ account for the difference in N peak positions betweenthese two materials. In addition, the S peak positions for the threesamples differ slightly from that for SLS. This suggests that LS alsomay be bonded differently in the samples than in SLS and may reflectbonding of the sulfate to the positively charged Lys tail amine. Thusthe N and S XPS peak position data for the three samples are consistentwith the formation of a new chemical bonding arrangement and supportformation of the Zn(Lys)₂(LS)₂ complex. Also, the Zn peak positions weresimilar for all three samples, suggesting Zn is primarily in the samechemical environment for each precipitate. Secondary Zn species were notdiscernable in the spectra.

TABLE 8 XPS N, S and Zn peak positions for samples 2A, 2B and 2C. XPSPeak, eV Sample N (1s) N⁺ (1s) S (2p) Zn (2p3/2) Sample 2A 399.7 401.5168.4 1021.8 Sample 2B 399.6 401.5 168.4 1021.8 Sample 2C 399.6 401.5168.5 1021.8 Lysine 399.0 400.9 — — SLS — — 168.7 —

To further understand the structure of a Zn-Lys-LS complex, ¹H NMRspectroscopy was applied to the sample 2A, 2B and 2C in methanol. ¹H NMRspectroscopy was performed as described in Example 1. Because thesesamples are not completely dissolved in methanol, the supernatant wassampled and analyzed by NMR. As shown in FIG. 7. ¹H NMR spectra of threesamples are identical in methanol and no structural differences aredetected. The chemical shifts corresponding to LS in samples 2A, 2B and2C do not show notable changes, implying that the interaction between LSand Zn, if present, is weak. In contrast, ¹H chemical shiftscorresponding to lysine in the three samples show detectable changes.Particularly, a proton peak in the complex displays a significant linebroadening and shifts from 3.55 ppm to 3.8 ppm, likely due to zincbinding. In fact, such exchange line-broadening has been observed inZn-Lysine complex (ZLC) in water. Moreover, based on the peak integralsin ¹H NMR spectra shown in FIG. 7, it is concluded that the mole ratiobetween LS and Lysine in samples 2A, 2B and 2C is 1:1.

¹³C NMR spectroscopy was further performed to analyze the molecularinteraction in these samples. ¹³C NMR spectra were acquired on a BrukerAvance III HD spectrometer (Bruker-Biospin, Billerica, Mass., USA) witha 5 mm BBI probe operating at 75.4 MHz at 25° C. Consistent with the ¹HNMR data, the carbonyl carbon of lysine at 174 ppm in sample 2A issignificantly broadened due to an exchange effect, supporting lysineinteraction with zinc. This result is in good agreement with the ¹H NMRfinding that a proton of lysine in this complex has exchange broadening.However, the chemical shifts corresponding to LS and lysine in sample 2Ado not show significant changes in ¹³C NMR spectrum. The chemical shiftdifference may be limited by the solubility of sample in methanol in ¹³CNMR spectroscopy.

To study the Zn(Lys)₂(LS)₂ complex structure further, LC-MS analysis wasperformed on samples 2A, 2B and 2C. LC-MS analysis was carried out asdescribed in Example 1. FIG. 8 and FIG. 9 show the mass spectrometrydata for samples 2A, 2B and 2C in methanol under positive and negativedetection modes. Because the complexes were not fully soluble inmethanol, supernatant was measured in all cases. MS under a positivedetection mode revealed several zinc species, including (Zn(Lys)₂)²⁺(at355 Da), (Zn(Lys)LS)⁺(at 475 Da), (Zn(Lys)₂LS) (at 621 Da), andZn(Lys)₂(LS)₂ (at 887 Da). This result is in agreement with theelemental analysis and spectroscopic data. The spectra of all threesamples are found to be similar with the exception that sample 2A doesnot display (Zn(Lys)₂)₂ ⁺ complex, likely due to its low intensity sincesample 2A exhibits the lowest MS signal among three samples. All speciescontaining LS appear as a cluster of peaks separated by 28 Dalton due toLS chain distribution (C₁₂ to C₁₈). Finally, as expected, free LS isidentified by MS under a negative detection mode (FIG. 9). The fourpeaks near 300 Da with mass difference of 28 Dalton correspond to freeLS and indicate the LS distribution with [(CH₂)₂] variation from C₁₂ toC₁₈.

Potentiometric titration is one of the most commonly used techniques todetermine metal-ligand binding constants (Martell et al. (1992),Determination and Use of Stability Constants, 2nd ed. Wiley. New York).The technique is based on the principle that hydrogen ion concentrationduring a pH-titration is sensitive to the protonation state and bindingconstant of ligands and metals in a given solution. By performingappropriate titration experiments and applying curve-fitting techniques,it is possible to calculate metal-ligand stability constants. A seriesof titration experiments were performed to determine the bindingconstants relevant to the zinc/lysine/SLS system. Titration experimentswere performed using a Metrohm Titrando 902 auto-titration unit with apH electrode. A solution of 0.25M KOH was freshly prepared in a 50:50mixture of glycerin:de-ionized water. Separately, solutions containingZn(NO₃)₂.6H₂O, SLS, KNO₃, and Lysine-HCl were prepared in a 50:50mixture of glycerin:de-ionized water. All solutions were prepared with aconcentration of 100 mM KNO₃ and 5 mM Zn. The concentrations ofLysine-HCl and SLS were adjusted to achieve various mole ratios ofZn:lysine:SLS. Prior to performing the titration experiments, eachsolution was adjusted to pH=1.0 using HNO₃. Titrations were conducted ina jacketed vessel with temperature maintained at 25 C. With rapidstirring, the 0.25M KOH was added to each test solution at a dosing rateof 0.5 mL/min until a final pH of 12 was achieved. The solution pH wasrecorded at 10 s intervals. 250 data points were collected for eachtitration. Titration curves were analyzed using SUPERQUAD to fit thestability constants. The best fit to the experimental data was obtainedby including a species formulated as Zn(HLys)₂(LS)₂ (FIG. 10A-C).Zn(HLys)₂(LS)₂, where lysine side-chain amino group is protonated atstudied pH conditions, is the same as Zn(Lys)₂(LS)₂ described above.Stability constants determined by potentiometric titration for the Zn,Lysine, SLS system is shown in Table 9.

TABLE 9 Stability constants determined by potentiometric titration forthe Zn, Lysine, SLS system. Complex Log(B) sigma HLys 10.07 — H₂Lys19.21 — H₃Lys 21.47 — HLS −2.0 — ZnHLys 14.68 — ZnH₂Lys₂ 28.34 — ZnLS1.84 0.47 ZnLS₂ 4.29 0.63 ZnHLysLS 16.96 0.17 ZnH₂Lys₂LS₂ 32.86 0.06The stability constant for Zn(HLys)₂(LS)₂ was determined aslog(B)=32.86. The stepwise stability constant for the reaction ofZn(HLys)₂ with two equivalents of SLS is log(K)=4.53. The relativelylarge stability constant for this complex indicates that formation ishighly favored. Speciation calculations (FIG. 10D) on a systemcontaining 0.1M Zn, 0.2M lysine, pH=8, and I=0.1M with a varyingconcentration of SLS indicate that SLS reacts nearly quantitatively withZn(HLys)₂ to form Zn(HLys)₂(LS)₂.

The Density Functional Theory (DFT) calculations were performed to checkwhether the formation of Zn-Lys-LS complex where LS is directly bound tozinc is energetically favorable. DFT calculations were done as describedin Example 1. The results are shown below.

ΔG⁰ (kcal/mol) K_(eq) [Zn(Lys)₂Cl]⁺(aq) + Ls⁻(aq) → 13.7 9.0 × 10⁻¹¹[Zn(Lys)₂Ls]⁺(aq) + Cl⁻(aq) [Zn(Lys)₂Cl]⁺(aq) + 2Ls⁻(aq) → 16.6 6.7 ×10⁻¹³ [Zn(Lys)₂Ls₂](aq) + Cl⁻(aq) [Zn(Lys)₂Cl]⁺(aq) + Ls⁻(aq) → 14.0 5.4× 10⁻¹¹ [ZnLysLsCl](aq) + Lys(aq) [Zn(Lys)₂Cl]⁺(aq) + Ls⁻(aq) → 20.4 1.1× 10⁻¹⁵ [ZnLysLs]⁺(aq) + Lys(aq) + Cl⁻(aq)The computational data demonstrate that each of the reactions abovewould be unfavorable. LS⁻ is so bulky that it causes a large sterichindrance and a low coordination number. In the example with two LSligands, the second LS ion is interacting with the NH₂ group on onelysine ligand via hydrogen bonding. In the input structure LS⁻ wascoordinated to Zn directly but shifted during the optimization due tosteric hindrance effects, thus favoring 5-coordination over6-coordination complex. In all four products LS⁻ acts as a monodentateligand without forming a chelate ring.

Example 3—Antibacterial Testing

The chelation of zinc can modify its efficacy as an antibacterial agent.To determine the antibacterial activity of zinc-amino acid-laurylsulfate complexes, experiments were conducted to measure the metabolicinhibition of S. mutans after treatment with Zn(NO₃)₂, ZLC,Zn(Lys)₂(LS)₂ (prepared in Example 2, sample 2C), [Zn(Arg)₂]²⁺,Zn(Arg)₂(LS)₂ (prepared in Example 1, sample 1B), and SLS. S. mutans isone of the prevalent bacteria present in the oral cavity. A concentratedsuspension of S. mutans in 135 mM KCl was treated with a knownconcentration of zinc salt to obtain a final zinc concentration of 2.5ppm. In the case of SLS, an equimolar amount of SLS was added to matchthe concentration of LS in the samples of Zn(Lys)₂(LS)₂. Using anautotitrator (Methrom Titrando 902) equipped with a 0.01M solution ofKOH, the bacterial suspension was equilibrated to pH=7. Afterequilibrating, glucose was added to the solution. The autotitratormaintained the pH at 7 through the precise addition of KOH solution. Theexperiment was allowed to proceed for 30 minutes and the volume of baseadded was recorded at 10 second intervals. After 30 mins, a growth curveof base addition over time was obtained. The area under this curve wascompared to the area under the curve for a control sample containingonly bacteria and used to calculate a % reduction value. The controlsample was analyzed from the same bacterial suspension as the testsample and the two samples were analyzed within 3 hours of each other.In order for data from a given suspension of S. mutans to be included,the control sample must consume at least 5 mL of base solution duringthe 30 minute time period. The % reduction for each sample is shown inTable 10.

TABLE 10 Reduction in the metabolic activity of S. mutans aftertreatment with various compounds. Sample % Metabolic Reduction Zn(NO₃)₂67.16 ± 2.49 Sodium lauryl sulfate 34.12 ± 1.56 ZLC 53.90 ± 2.36Zn(Lys)₂(LS)₂ 67.19 ± 3.97 [Zn(Arg)₂]²⁺ 67.19 ± 0.32 Zn(Arg)₂(LS)₂ 72.53± 3.76

The antibacterial testing results show that despite low solubility,Zn(Lys)₂(LS)₂ shows a better antibacterial performance compared to ZLC.Similarly Zn(Arg)₂(LS)₂ shows a directionally improved antibacterialperformance compared to [Zn(Arg)₂]²⁺.

While the disclosure has been described with respect to specificexamples including presently preferred modes of carrying out thedisclosure, those skilled in the art will appreciate that there arenumerous variations and permutations of the above described systems andtechniques. It is to be understood that other embodiments may beutilized and structural and functional modifications may be made withoutdeparting from the scope of the present disclosure. Thus, the scope ofthe disclosure should be construed broadly as set forth in the appendedclaims.

The invention claimed is:
 1. A zinc-amino acid-lauryl sulfate complex,wherein the complex is in powder form.
 2. The complex of claim 1,wherein the zinc-amino acid-lauryl sulfate has the chemical structure[Zn(amino acid)₂](LS)₂.
 3. The complex of claim 1, wherein the complexis water insoluble.
 4. The complex of claim 1, wherein the amino acid isa basic amino acid.
 5. The complex of claim 4, wherein the basic aminoacid is arginine or lysine.
 6. The complex of claim 5, wherein the basicamino acid is arginine and the complex has the chemical structure[Zn(Arg)₂](LS)₂.
 7. The complex of claim 5, wherein the basic amino acidis lysine and the complex has the chemical structure [Zn(Lys)₂](LS)₂. 8.An oral care composition comprising the zinc-amino acid-lauryl sulfatecomplex according to claim 1, wherein the zinc-amino acid-lauryl sulfatecomplex is added to the composition as a preformed complex.
 9. The oralcare composition of claim 8, wherein the zinc-amino acid-lauryl sulfatecomplex is present in an amount of 0.1 to 10% by weight of thecomposition.
 10. The oral care composition of claim 8, wherein the oralcare composition is a dentifrice.
 11. The oral care composition of claim8, wherein the oral care composition comprises a fluoride ion source.12. A method of producing the zinc-amino acid-lauryl sulfate complexaccording to claim 1, comprising combining sodium lauryl sulfate and azinc-amino acid complex in an aqueous solution.
 13. The method of claim12, wherein the zinc-amino acid-lauryl sulfate complex is a zinc-basicamino acid-lauryl sulfate complex.
 14. The method of claim 13, whereinthe zinc-amino acid complex is [Zn(Lys)₂Cl]⁺Cl⁻ (ZLC) or[Zn(Arg)₂Cl]⁺Cl⁻.
 15. The method of claim 13, wherein the zinc-aminoacid complex is prepared by combining a soluble zinc salt and a basicamino acid, e.g., arginine or lysine, in an aqueous solution.
 16. Themethod of claim 12, wherein the method further comprises a step offiltering and drying the zinc-amino acid-lauryl sulfate complex.